The major histocompatibility (MHC) antigens are cell surface glycoproteins with central roles in the generation of immune responses. The class I molecules, which were originally described as the classical transplantation antigens involved in allograft tissue rejection, also present antigens from virally infected, chemically modified, and tumor cells to cytotoxic T cells (CTL). Proper recognition of class I complexes in both alloreactive and MHC-restricted responses is critical for the initiation of T cell mediated- responses. In addition to the role of MHC proteins in the initiation of T cell-mediated allospecific and MHC-restricted responses, class I proteins also transmit transmembrane signals. The goals of the proposed studies are to analyze the structural characteristics of MHC molecules required in both the generation of allospecific responses as well as in the transmission of transmembrane signals. Preliminary work suggests that the region on the class I H-2Kb molecule between amino acid residues 163-174 serves as a docking site on the MHC molecule for T cell receptors. To analyze the extent and critical interactions between this region of the MHC molecule and T cell receptors, nested peptides spanning this region will be tested for their ability to block T cell activation and single amino acid mutations will be inserted into H-2Kb between amino acid residues 163-174, the region of the H2Kbm10 mutation. Experiments to study additional T cell contact regions from areas of exposed alpha helix will analyze the ability of other peptides to block alloreactive T cells. To study the functional and biochemical interactions between MHC molecules and T cell receptors, soluble analogs of class I proteins will be used. Soluble forms of both the wild type H-2Kb molecule and several of the closely related naturally occurring H-2Kbm mutant proteins, will be prepared. The soluble analogs, which will consist of both monomeric class I proteins and of a hybrid MHC molecule linked to an immunoglobulin heavy chain, will be used to study the concentration dependence and valency requirement of T cell activation. The binding properties of the soluble MHC analogs to clonotypic T cell receptors will also be characterized. Another apparent function of class I antigens is to transduce transmembrane signals. To study signal transduction mediated via the crosslinking of cell surface MHC molecules, cells expressing mutations in the intracytoplasmic protein domains (encoded by exons VI, VII, and VIII) of the class I molecule will be analyzed. The specific role of the phosphorylation site in exon VII will also be addressed. The studies outlined will further our understanding of the multi-faceted interactions between MHC proteins and T cells and the intrinsic role of MHC molecules in signal transduction. An enhanced understanding of these interactions will improve our understanding of the role of MHC antigens in the generation of both normal and pathological immune responses.